Metabolomics of developmental changes in Triatoma sanguisuga gut microbiota

Triatoma sanguisuga is one of the major vectors of Trypanosoma cruzi in the southeastern US, where it sustains a robust zoonotic parasite transmission cycle and occasional human infections. A better understanding of triatomine development may allow for alternative approaches to insecticide-based vector control. Indeed, the role of the gut microbiota and bacterial endosymbionts in triatomine development and in their vectorial capacity is emerging. We investigated here the differences in microbiota among nymph and adult T. sanguisuga, to shed light on the metabolomic interactions occurring during development. Microbiota composition was assessed by 16s gene amplification and deep sequencing from field-caught adult bugs and their laboratory-raised progeny. Significant differences in microbiota bacterial diversity and composition were observed between nymphs and adults. Laboratory-raised nymphs showed a higher taxonomic diversity, and at least seven families predominated. On the other hand, field-caught adults had a lower bacterial diversity and four families comprised most of the microbiota. These differences in compositions were associated with differences in predicted metabolism, with laboratory-raised nymphs microbiota metabolizing a limited diversity of carbon sources, with potential for resource competition between bacterial families, and the production of lactic acid as a predominant fermentation product. On the other hand, field-caught adult microbiota was predicted to metabolize a broader diversity of carbon sources, with complementarity rather than competition among taxa, and produced a diverse range of products in a more balanced manner. The restricted functionality of laboratory-raised nymph microbiota may be associated with their poor development in captivity, and further understanding of the metabolic interactions at play may lead to alternative vector control strategies targeting triatomine microbiota.


Introduction
Chagas disease is a parasitic illness which afflicts 6-8 million people in Latin America alone [1], and an estimated 288,000 people in the United States [2]. It is caused by Trypanosoma cruzi parasites, which are transmitted primarily by triatomine insect vectors, although additional transmission routes contribute to disease epidemiology. At least 11  can transmit T. cruzi in the United States, with Triatoma sanguisuga the most relevant species in the southeastern part of the country [3]. It is responsible for an active zoonotic parasite transmission cycle involving multiple sylvatic and synanthropic mammalian hosts, with frequent spill-over in domestic animals including pet dogs and cats [4][5][6][7], and occasional cases of autochthonous human cases, as seen in Mississippi [8], Tennessee [9] and Louisiana [10]. Vector control conventionally relies on residual insecticide spraying, which shows limitation in the case of intrusive sylvatic vectors [11], and alternative approaches are critically needed. In that sense, triatomine gut microbiota and the key contribution of specific bacterial endosymbionts to bug development and to its vectorial capacity are emerging as attractive targets for novels strategies. For example, the successful development of Rhodnius prolixus, one of the most epidemiologically relevant vector species in large parts of South America [12,13], is thought to be critically dependent on the provision of B-complex vitamins by its endosymbiont Rhodococcus rhodnii, which is acquired during bug development through coprophagy [14]. Thus, vitamin B deficiency in laboratory colonies leads to growth retardation and nymphs fail to reach the adult stage in the absence of R. rhodnii, although a more recent study suggests that additional bacteria in the gut microbiota may also contribute to vitamin B production [15]. Furthermore, other studies suggest that the microbiota is likely providing multiple metabolites in addition to vitamin B [16].
In spite of these studies, our understanding of the contribution of the microbiota to triatomine development remains limited. A general trend in field-caught bugs is that nymphs have a highly diverse microbiota, which often differs from that of adult bugs, suggesting different metabolic functions and interactions. This is the case for Triatoma protracta [17], Triatoma reticularia [17], Triatoma rubida [17], Triatoma dimidiata [18,19], and Rhodnius prolixus [20]. On the other hand, in Triatoma sordida the adult microbiota is more diverse compared to nymphs, with unique bacteria being incorporated from the 4 th instar stage onwards [21]. Furthermore, it is still unclear how such changes in microbiota composition may translate into differences in metabolic profiles and interactions among bacteria and the bugs. In T. sanguisuga, too limited data is available to assess changes in microbiota during development [17], but this species is notoriously difficult to raise in captivity as nymphs mostly stop molting in the third instar stage and die [22,23]. This has been associated with the requirement of maintaining nymphs together with field-collected adults, under the hypothesis that this allowed them to acquire necessary flora through coprophagy [23].
The gut microbiota also likely interacts with T. cruzi parasites as these reside in the triatomine gut, and the microbiota may modulate vectorial capacity as bacteria and parasite compete for metabolites and may inhibit one another. For example Serratia marcescens, another member of R. prolixus microbiota, has a trypanolytic activity that can antagonize T. cruzi growth [24,25]. In fact, T. cruzi infection leads to changes in microbiota composition in several triatomine species, suggesting tight interactions among bacteria and the parasite [26][27][28][29]. These interactions may also be highly parasite strain-specific, as some differences in microbiota composition were detected according to T. cruzi parasite DTU present in T. sanguisuga [4] and T. dimidiata [29]. The introduction of transgenic organisms into the microbiota of triatomine vectors through various strategies has been proposed before as a potential approach to promote the production of trypanolytic factors by endosymbionts to reduce T. cruzi establishment in vectors, hence reducing vector capacity [30,31], and a better understanding on microbiota functions may lead to effective targets.
Because T. sanguisuga may be an interesting model to better evaluate the role of the microbiota in triatomine development, we sought to assess potential differences in microbiota between nymphs and adults, to test the hypothesis that laboratory-raised nymphs lack key bacteria compared to field-collected adults [23]. We further predict key functional metabolic properties of the identified microbiota as a first step towards evaluating microbiota metabolic functions in T. sanguisuga.

Triatomines and DNA extraction
Insects were collected across Louisiana, USA, through community participation, in the years 2019 and 2021, and brought to an biosafety level 2 facility for diagnosis of T. cruzi in the insect feces via PCR. Uninfected adult females were kept individually in Nalgene containers with mesh lined tops and filter paper folds to lay eggs. Bugs were fed every 2-3 weeks via bell feeder using sheep blood. Eggs and hatched nymphs were kept in the same containers with the adults. All nymphs failed to molt past the third instar stage and died. All bugs were collected and stored at -20ºC until used. A total of 23 insects were used: 18 nymphs (8 first instar, 6 second instar, and 4 third instar) and 5 female adults. For adults, DNA was extracted from the distal section of insect abdomens using a sterile disposable scalpel cleaned with dilute bleach, whereas for nymph the entire insect was used. DNA was extracted using the Qiagen DNEasy blood and tissue extraction kit following manufacturer's instructions.

16s RNA gene amplification and sequencing
The full-length 16s RNA gene (about 1500 bp) was amplified using Oxford Nanopore™ 16s Barcoding kit (SQKRAB204) following the manufacturer's instructions except that two rounds of PCR amplification were performed for library preparation. DNA libraries were sequenced on a Nanopore MinIon platform. Raw sequence data are available in the NCBI SRA database under BioProject PRJNA892946, BioSamples SAMN31403430-.SAMN31403445.

Sequence and data analysis
Fastq DNA sequences were filtered for quality and length and analyzed with a Bayesian classifier from the Ribosomal Database Project [32] as implemented in Geneious Prime. We used a threshold of �97% sequence identity for taxonomic identification of bacterial taxa at the family level. Microbiota composition was then analyzed using MicrobiomeAnalyst [33,34]. Data were first rarefied and normalized using the Total Sum Scaling (TSS) method, to account for variability in sequencing depth. Chao1 and Shannon alpha diversity indices were calculated and compared between groups using t-tests. For beta diversity, Non-Metric Dimensional Scaling (NMDS) was used based on Bray-Curtis distances and statistical significance of differences was assessed by Permutational ANOVA (PERMANOVA). Associations of individual bacterial families with triatomine developmental stage were assessed by correlation analysis. A cooccurrence network of bacterial families based on SparCC correlations was also constructed to identify bacteria which presence/absence are strongly correlated within a specific developmental stage of T. sanguisuga. FDR corrections were used to account for multiple testing in establishing the statistical significance of the associations.
For metabolic analysis, the AGORA database from the Virtual Metabolic Human was used, which provides metabolic reconstruction of over 800 bacterial species from microbiomes [35]. Carbon sources and fermentation products associated with the most abundant bacterial families identified in T. sanguisuga were extracted and weighted based of the proportion of species within each family able to use/produce each metabolite. Seven bacterial families were included for nymphs, and four for adults, which accounted for over 85% of the microbiota for each group. The relative level of each metabolite used/produced by each bacterial family was calculated based on their relative abundance in the microbiota, to establish the predicted integrated metabolic profile of the bacterial community.

Microbiota composition of nymph and adult T. sanguisuga
A total of 23 bugs were included in the study, but data from seven first instars were discarded from the analysis due to poor DNA extraction, 16s amplification or sequencing. The remaining 16 samples yielded an average of 80,947 reads per bug. After filtering and normalization, rarefaction curves indicated that this sequencing amount was sufficient to recover most of the bacterial diversity of these samples (S1 Fig). An initial comparison of second and third instar microbiota composition indicated that their alpha and beta diversity were not significantly different (S2 Fig), and these were combined into a single nymph group for further comparison with adult microbiota.
Comparison of alpha diversity between T. sanguisuga laboratory-raised nymph and fieldcaught adult microbiota indicated that nymphs harbored a greater diversity of bacterial families than adults, as indicated by Shannon and Chao1 indices, although the later did not reach statistical significance (Fig 1A and 1B, t = 2.88, P = 0.017 and t = 0.29, P = 0.78, respectively). Indeed, laboratory-raised nymph's microbiota included up to 14 bacterial families, while only 10 were detected in field-caught adults (Fig 1C). Seven taxa comprised over 85% of the laboratory-raised nymph microbiota, including Xanthomonadaceae, Moraxellaceae, Clostridales incertae sedis, Propionibacteriaceae, Staphylococcaceae, Burkholderiales incertae sedis and Aerococcaceae, while the field-caught adult microbiota included four predominant taxa: Enterobacteriaceae, Porphyromonadaceae, Staphylococcaceae and Peptoniphilaceae. Analysis of beta diversity confirmed that there was a significant difference in microbiota composition between nymph and adult bugs (Fig 2, F  Four families rather abundantly present in laboratory-raised nymphs were absent in fieldcaught adults (Burkholderiales incertae sedis, Carnobacteriaceae, Comamonadaceae and Moraxellaceae), and an additional six families present in both nymphs and adults were strongly decreased in adults (Aerococcaceae, Burkholderiaceae, Clostridales incertae sedis, Propionibacteriaceae, Streptococcaceae and Xanthomonadaceae). On the other hand, Enterobacteriaceae, Porphyromonadaceae, Staphylococcaceae, which were minor component of the laboratory-raised nymph microbiota, were strongly increased and predominated in the microbiota of field-caught adults.

Associations among bacterial families and developmental stage
To further assess which bacteria may associate with specific developmental stage, we performed correlation analysis. Peptoniphilaceae, Porphyromonadaceae and to a lesser extent Enterobacteriaceae were significantly associated with the field-caught adult microbiota, while Carnobacteriaceae, Clostridales incertae sedis, and Streptococcaceae were significantly associated with laboratory-raised nymph microbiota (Fig 3). Analysis of co-occurrence networks also indicated that several bacterial families were occurring simultaneously in a microbiota, suggesting functional interactions. For example, Peptoniphilaceae and Porphyromonadaceae were frequently found together in field-caught adults, while Propionibacteraceae and Clostridales insertae sedis co-occurred in laboratory-raised nymphs (Fig 4). Similarly, Moraxellaceae, Burkholderiales incertae sedis and Comamonadaceae co-occured in nymphs and may antagonize Staphylococcaceae which was more abundant in adults.

Metabolic function of T. sanguisuga microbiota
The contribution of the predominant bacterial families to the integrated microbiota metabolic functions was then predicted, based on the AGORA database [35]. We included metabolic functions from seven families in nymphs and four in adults, which represented over 85% of their respective microbiota. Bacteria from the laboratory-raised nymph microbiota used a limited variety of carbon sources, in which glucose largely predominated, and the use of the same carbon sources by multiple bacterial families suggested potential competition for these resources (Fig 5A). The fermentation products of the laboratory-raised nymph microbiota included mostly lactic and acetic acid, and a limited variety of additional compounds in lesser

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amounts (Fig 5B). On the other hand, the field-caught adult microbiota, in spite of comprising a limited diversity of bacteria, appeared able to process a broader diversity of carbon sources in a more balanced manner, even though glucose was also an important source (Fig 5C). Also, while the adult microbiota fermentation products included lactic and acetic acids, it was not as biased as that of laboratory-raised nymphs, and a larger diversity of compounds were also produced in more comparable relative amounts (Fig 5D). For example, indole, (2R,3R)-2,3-butanediol and phenylacetic acid were only produced by the adult microbiota. Together, these data point to important differences in metabolic functions of the microbiota between laboratoryraised nymphs and field-caught adults, with the microbiota from adults presenting potential functional gains in terms of metabolic properties.

Discussion
Triatomine gut microbiota is thought to play a key role in bug metabolism by providing key functions and metabolites to maximize the use of blood meals from these strict hematophagous bugs. However, the role of the microbiota in triatomine development remains poorly understood, in particular for T. sanguisuga, for which deficiencies in endosymbionts have been proposed to explain the poor survival of nymphs in captivity [23]. Our comparison of microbiota between laboratory-raised nymphs and field-caught adults provides initial support to this hypothesis and underlines metabolic differences that may explain the limited survival of nymphs in these conditions. Indeed, our data clearly show that there is no/limited vertical transmission of the field-caught adult microbiota to their laboratory-raised offspring, suggesting that nymphs nymphs need to acquire bacteria from their environment, and the ones they acquire under laboratory condition are insufficient for their development. Thus, the microbiota of T. sanguisuga seems to follow the general trend observed in other species of triatomines, with a more complex and diverse microbiota present in nymphs, while the adult microbiota is largely dominated by fewer taxa [17,18,20].
These data suggest a major reshuffle of the microbiota during development, with most taxa present in nymphs being replaced by a more limited set of taxa in adults, likely through coprophagy. However, at the functional level, the simpler adult microbiome of T. sanguisuga actually appears to provide a more complex metabolic network. The simplification of the microbiota in adults thus results in significant gains in metabolic functions. Indeed, the microbiota from laboratory-raised nymphs appears limited in its metabolic capabilities, with potential for competition among taxa, which may result in an unstable bacterial community of limited value to the bugs, leading to poor growth. On the other hand, the reduced complexity of the field-caught adult microbiota may allow for less competition, while at the same time ensuring a greater diversity of metabolic functions, which may better maximize the use of blood metabolites. However, it remains difficult to assess what key metabolic function(s) is/are provided by the filed-caught adult microbiota that is/are absent in the laboratory-raised nymph microbiota based on our predicted data. In Rhodnius prolixus, the supplementation of blood with vitamin B compounds can in part compensate for the absence of Rhodococcus rhodnii endosymbionts in laboratory-raised bugs [36]. It is however unclear what key metabolic function Peptoniphilaceae, Porphyromonadaceae and Enterobacteriaceae, which predominate in adult T. sanguisuga, may be providing to the bug. However, given the magnitude of the differences in bacterial taxa between nymphs and adults, it is likely that these bacteria are involved in multiple metabolic functions.
Triatomines also emerge as rather different from other Hemiptera, which as hemimetabolous insects are considered to have a rather stable microbiota during development [37]. Indeed, a dramatic reorganization of the gut microbiota during development has been proposed as a key feature of holometabolous insects, due to the major internal reconstructions during the pupal stage, which includes the replacement of the gut epithelium, while this process does not occur in hemimetabolous insects [37]. In that sense, the strict blood feeding of triatomine likely poses unique metabolic challenges, that may require more specific functions from their microbiota, hence more changes during bug development, which may be unique among Hemiptera and more generally in hemimetabolous insects.
There are however some limitations in our study. First, our limited sample size and the failure of bugs to molt past the third stage precluded an analysis through all laboratory-raised

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nymphal stages and adults, as changes may be progressive as seen in R. prolixus [20], or more rapid. Also, only female adults were included, but previous work did not find significant differences between male and female microbiota in T. sanguisuga [4]. The inclusion of field-collected T. sanguisuga nymphs would also expand our analysis by providing further information on how these changes in microbiota occur in natural conditions. Finally, further refinement of The relative abundance of carbon sources metabolized by nymph microbiota (A) is compared to those from the adult microbiota (B). Similarly, the relative amounts of fermentation products from the nymph microbiota (C) are compared with those from the adult microbiota (D). The relative contribution of the indicated bacterial taxa in the use/ production of metabolites is color-coded as indicated.
https://doi.org/10.1371/journal.pone.0280868.g005 the microbiota at the species level would allow for more detailed metabolic predictions, as there is some heterogeneity in metabolic routes within bacterial families. Also, dissecting and analyzing different segments of the digestive tract of the bugs would allow further assessing potential differences in microbiota composition along the gut and the role of metabolic compartmentalization.
In conclusion, our study shows that T. sanguisuga nymphs with developmental failure have a diverse but metabolically limited microbiota, very different from that of field-collected adults. The adult microbiota, while including a lower bacterial diversity, provides a broader metabolic capacity that may be better suited to triatomine blood feeding diet. Further analysis, including of additional triatomine species, is warranted, as well as more detailed metabolic reconstructions of their microbiota for a better understanding of its role in triatomine biology and development.